Covalent binding to tubulin by isothiocyanates - A mechanism of cell growth arrest and apoptosis
Covalent binding to tubulin by isothiocyanates - A mechanism of cell growth arrest and apoptosis
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DOI：
10.1074/jbc.m802330200
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发表时间：
2008-08-08
影响因子：
4.8
通讯作者：
Chung, Fung-Lung
中科院分区：
文献类型：
--
作者：
Mi, Lixin;Xiao, Zhen;Chung, Fung-Lung
在十字花科蔬菜中发现的异硫氰酸酯（ITC），包括苄基-ITC（BITC）、苯乙基-ITC（PEITC）和萝卜硫素（SFN），在动物模型中抑制致癌作用并在各种细胞类型中诱导凋亡和细胞周期停滞。ITC抑制细胞生长的生化机制尚未完全了解。我们最近的研究表明，ITC与细胞内蛋白的结合可能是诱导细胞凋亡的重要起始事件。然而，具体的蛋白质靶点和分子机制尚未确定。在这项研究中，二维凝胶电泳的人肺癌A549细胞处理与放射性标记的PEITC和SFN显示，微管蛋白可能是一个主要的在体内结合的目标ITC。我们研究了ITCs与微管蛋白的结合是否会导致细胞生长停滞。ITCs对A549细胞的增殖有明显的抑制作用，其抑制作用大小顺序为BITC > PEITC > SFN。所有三个ITC也诱导有丝分裂停滞和细胞凋亡的活性相同的顺序。我们发现，ITCs破坏微管聚合在体外和体内具有相同的顺序的效力。质谱分析表明微管蛋白中的半胱氨酸被ITCs共价修饰。Ellman分析结果表明，修饰水平遵循相同的顺序，BITC > PEITC > SFN。总之，这些结果支持微管蛋白是ITC的靶点并且ITC-微管蛋白相互作用可导致下游生长抑制的观点。这是第一项直接将微管蛋白-ITC加合物形成与细胞生长抑制联系起来的研究。
Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.